Antibodies to quetiapine haptens and use thereof

ABSTRACT

Disclosed is an antibody which binds to quetiapine, which can be used to detect quetiapine in a sample such as in a competitive immunoassay method. The antibody can be used in a lateral flow assay device for point-of-care detection of quetiapine, including multiplex detection of aripiprazole, olanzapine, quetiapine, and risperidone in a single lateral flow assay device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/971,546, filed Aug. 20, 2013, and published Feb. 27, 2017, as US2014/0057305, which claims the benefit of U.S. Provisional ApplicationNo. 61/691,598, filed Aug. 21, 2012, all of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of immunoassays, and inparticular to antibodies that bind to quetiapine which can be used inimmunoassays for detection of quetiapine.

BACKGROUND

Schizophrenia is a chronic and debilitating psychiatric disorderaffecting approximately 0.45-1% of the world's population (van Os, J.;Kapur, S. “Schizophrenia” Lancet 2009, 374, 635-645). The principalgoals of treatment are to achieve sustained remission from psychoticsymptoms, reduce the risk and consequences of relapse, and improvepatient functioning and overall quality of life. While many patientswith schizophrenia are able to achieve symptom stability with theavailable antipsychotic medications, poor adherence to medication is acommon reason for relapse with daily administered oral medications.Several studies (Abdel-Baki, A.; Ouellet-Plamondon, C.; Malla, A.“Pharmacotherapy Challenges in Patients with First-Episode Psychosis”Journal of Affective Disorders 2012, 138, S3-S14) investigating theoutcomes of non-compliance have shown that patients with schizophreniawho do not take their medication as prescribed have higher rates ofrelapse, hospital admission and suicide as well as increased mortality.It is estimated that 40 to 75% of patients with schizophrenia havedifficulty adhering to a daily oral treatment regimen (Lieberman, J. A.;Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins,D. O.; Keefe, R. S. E.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.;Severe, J.; Hsiao, J. K. “Effectiveness of Antipyschotic Drugs inPatients with Chronic Schizophrenia” New England Journal of Medicine2005, 353(12), 1209-1223).

Therapeutic drug monitoring (TDM) is the quantification of serum orplasma concentrations of drugs, including anti-psychotic drugs, fortreatment monitoring and optimization. Such monitoring permits, forexample, the identification of patients that are not adhering to theirmedication regimen, that are not achieving therapeutic doses, that arenon-responsive at therapeutic doses, that have suboptimal tolerability,that have pharmacokinetic drug-drug interactions, or that have abnormalmetabolism resulting in inappropriate plasma concentrations.Considerable individual variability exists in the patient's ability toabsorb, distribute, metabolize, and excrete anti-psychotic drugs. Suchdifferences can be caused by concurrent disease, age, concomitantmedication or genetic peculiarities. Different drug formulations canalso influence the metabolism of anti-psychotic drugs. TDM permits doseoptimization for individual patients, improving therapeutic andfunctional outcomes. TDM further permits a prescribing clinician toensure compliance with prescribed dosages and achievement of effectiveserum concentrations.

To date, methods for determining the levels of serum or plasmaconcentrations of anti-psychotic drugs involve the use of liquidchromatography (LC) with UV or mass spectrometry detection, andradioimmunoassays (see, for example, Woestenborghs et al., 1990 “On theselectivity of some recently developed RIA's” in Methodological Surveysin Biochemistry and Analysis 20:241-246. Analysis of Drugs andMetabolites, Including Anti-infective Agents; Heykants et al., 1994 “ThePharmacokinetics of Risperidone in Humans: A Summary”, J Clin Psychiatry55/5, suppl:13-17; Huang et al., 1993 “Pharmacokinetics of the novelanti-psychotic agent risperidone and the prolactin response in healthysubjects”, Clin Pharmacol Ther 54:257-268). Radioimmunoassays detect oneor both of risperidone and paliperidone. Salamone et al. in U.S. Pat.No. 8,088,594 disclose a competitive immunoassay for risperidone usingantibodies that detect both risperidone and paliperidone but notpharmacologically inactive metabolites. The antibodies used in thecompetitive immunoassay are developed against a particular immunogen. IDLabs Inc. (London, Ontario, Canada) markets an ELISA for olanzapine,another anti-psychotic drug, which also utilizes a competitive format.The Instructions For Use indicate that the assay is designed forscreening purposes and intended for forensic or research use, and isspecifically not intended for therapeutic use. The Instructionsrecommend that all positive samples should be confirmed with gaschromatography/mass spectrometry (GC-MS), and indicate that the antibodyused detects olanzapine and clozapine (see ID Labs Inc., “InstructionsFor Use Data Sheet IDEL-F083”, Rev. Date Aug. 8, 2011). Some of thesemethods, namely HPLC and GC/MS, can be expensive and labor-intensive,and are generally only performed in large or specialty labs having theappropriate equipment.

A need exists for other methods for determining the levels ofanti-psychotic drugs, particularly methods that can be performed in aprescribing clinician's office (where the treatment for an individualpatient can be adjusted accordingly in a much more timely manner) and inother medical settings lacking LC or GC/MS equipment or requiring rapidtest results.

Quetiapine is:

SUMMARY OF THE INVENTION

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to quetiapine and which: (i) is generatedin response to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i).

Formula I:

wherein:R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H, or, Z—(Y)_(p)-G;R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -alkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;wherein:W is selected from the group consisting of:—C(O)—, alkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

Presently preferred embodiments of the antibody of the subject inventionare the antibodies designated 11, 89-3, 89-5, and 89-13 generatedagainst the compound having Formula II. Another suitable immunogen isthe compound having Formula III.

Formula II (Compound 9):

Formula III (Compound 8):

The antibodies of the subject invention can be provided in assay kitsand assay devices, with a presently preferred device being a lateralflow assay device which provides for point-of-care analysis.

The invention further provides a method of producing an antibody whichbinds to quetiapine, the method comprising: (i) selecting a host cellfor antibody production; and (ii) inoculating the host with a conjugateof a compound of Formula I and an immunogenic carrier, wherein the hostproduces an antibody which binds to quetiapine. Further provided is amethod of producing a hybridoma cell line capable of producing amonoclonal antibody which binds to quetiapine. The method comprises: (i)selecting a host for antibody production; (ii) inoculating the host witha conjugate of a compound of Formula I and an immunogenic carrier; (iii)fusing a cell line from the inoculated host with a continuously dividingcell to create a fused cell capable of producing a monoclonal antibodywhich binds to quetiapine; and (iv) cloning the fused cell so as toobtain a hybridoma cell line.

The invention further provides a method of detecting quetiapine in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and quetiapine present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect quetiapine in the sample.

Further provided is a competitive immunoassay method for detectingquetiapine in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and with quetiapineor a competitive binding partner of quetiapine, wherein one of theantibody and the quetiapine or competitive binding partner thereof islabeled with a detectable marker, and wherein sample quetiapine competeswith the quetiapine or competitive binding partner thereof for bindingto the antibody; and (ii) detecting the label so as to detect samplequetiapine.

Further objects, features and advantages of the present invention willbe apparent to those skilled in the art from detailed consideration ofthe preferred embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show Competitive ELISA results generated with varioushybridomas;

FIG. 3 shows the competitive immunoassay format used on a lateral flowassay device;

FIG. 4 shows a typical dose response curve generated with quetiapinesub-clones 89-3, 89-13, and 89-5;

FIG. 5 shows the chip design of a lateral flow assay device according tothe subject invention;

FIG. 6 shows a typical dose response curve for an aripiprazole positivecontrol generated with antibody 5C7 and a labeled aripiprazolecompetitive binding partner;

FIG. 7 shows a typical dose response curve for an olanzapine positivecontrol generated with antibody 4G9-1 and a labeled olanzapinecompetitive binding partner;

FIG. 8 shows a typical dose response curve for a quetiapine positivecontrol generated with antibody 11 and a labeled quetiapine competitivebinding partner;

FIG. 9 shows a typical dose response curve for a risperidone positivecontrol generated with antibody 5-9 and a labeled risperidonecompetitive binding partner;

FIG. 10 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence oflabeled aripiprazole competitive binding partner, with no dose responsecurve for olanzapine, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 11 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 12 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence of alabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 13 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence of alabeled competitive binding partner for each;

FIG. 14 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence ofa labeled aripiprazole competitive binding partner, with no doseresponse curve for olanzapine, quetiapine, or risperidone in thepresence of antibody and labeled competitive binding partner for each;

FIG. 15 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 16 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence oflabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 17 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence ofantibody and labeled competitive binding partner for each;

FIG. 18 shows a comparison of the aripiprazole dose response curvegenerated as a positive control to the aripiprazole dose response curvegenerated in the multiplex format;

FIG. 19 shows a comparison of the olanzapine dose response curvegenerated as a positive control to the olanzapine dose response curvegenerated in the multiplex format;

FIG. 20 shows a comparison of the quetiapine dose response curvegenerated as a positive control to the quetiapine dose response curvegenerated in the multiplex format; and

FIG. 21 shows a comparison of the risperidone dose response curvegenerated as a positive control to the risperidone dose response curvegenerated in the multiplex format.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides an isolated antibody which binds to quetiapine.The invention further provides an assay kit and an assay devicecomprising the antibody. Also provided are methods of producing theantibody and of producing a hybridoma cell line capable of producing theantibody. Further provided is a method of detecting quetiapine in asample, including a competitive immunoassay method.

In one embodiment, the present invention is directed to an isolatedantibody or a binding fragment thereof, which binds to quetiapine andwhich: (i) is generated in response to a conjugate of a compound ofFormula I and an immunogenic carrier; or (ii) competes for an epitopewhich is the same as an epitope bound by the antibody of (i).

Formula I:

whereinR¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H, or, Z—(Y)_(p)-G;R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -alkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;wherein:W is selected from the group consisting of:—C(O)—, alkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H, or Z(Y)_(p)G;R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H, or, Z—(Y)_(p)-G;R³ is H, provided that either R¹ or R² must be H, and further providedthat both R¹ and R² may not be H simultaneously;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -alkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or CH₂NH—(Y)_(p)-G;

R² is H, or CH₂NH—(Y)_(p)-G; provided that either R¹ or R² must be H,and further provided that both R¹ and R² may not be H simultaneously;

R³ is H;

wherein:

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 1.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must beH, and further provided that both R¹ and R² may not be H simultaneously;R³ is H;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H,

or CH₂NH₂;R² is H,

or CH₂NH₂; provided that either R¹ or R² must be H, and further providedthat both R¹ and R² may not be H simultaneously;R³ is H;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula IV and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula V and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula VI and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toquetiapine and which: (i) is generated in response to a conjugate of acompound of Formula VII and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

Preferably, the antibody of the subject invention is generated inresponse to a conjugate of a compound selected from the compounds of:Formula I, Formula IV, Formula V, Formula VI, and Formula VII; and animmunogenic carrier.

Further details of the compounds described by the formulas above and theconjugates formed by the compounds and an immunogenic carrier areprovided in the section below entitled “Compounds, Conjugates andImmunogens”.

Further details of the antibodies of the subject invention are providedin the section below entitled “Antibodies”.

The subject invention further provides an assay kit comprising theantibody, as well as an assay device comprising the antibody.Preferably, the assay device is a lateral flow assay device. Furtherdetails of the assay kits and assay devices are provided below in thesection entitled “Assay Kits and Devices”.

The invention further provides a method of producing an antibody whichbinds to quetiapine, the method comprising: (i) selecting a host cellfor antibody production; and (ii) inoculating the host with a conjugateof a compound of Formula I and an immunogenic carrier, wherein the hostproduces an antibody which binds to quetiapine. In additionalembodiments, the conjugate used in the method can be a conjugate of acompound selected from the compounds of: Formula IV, Formula V, FormulaVI, and Formula VII; and an immunogenic carrier. Further details on theproduction of the antibodies of the subject invention are provided inthe section below entitled “Antibodies”.

Further provided is a method of producing a hybridoma cell line capableof producing a monoclonal antibody which binds to quetiapine. The methodcomprises: (i) selecting a host for antibody production; (ii)inoculating the host with a conjugate of a compound of Formula I and animmunogenic carrier; (iii) fusing a cell line from the inoculated hostwith a continuously dividing cell to create a fused cell capable ofproducing a monoclonal antibody which binds to quetiapine; and (iv)cloning the fused cell so as to obtain a hybridoma cell line. Inadditional embodiments, the conjugate used in the method can be aconjugate of a compound selected from the compounds of: Formula IV,Formula V, Formula VI, and Formula VII; and an immunogenic carrier.Further details of the production of hybridomas in accordance with thesubject invention are provided in the section below entitled“Antibodies”.

The invention further provides a method of detecting quetiapine in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and quetiapine present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect quetiapine in the sample. Further details of the method ofdetecting quetiapine in accordance with the subject invention areprovided in the section below entitled “Immunoassays”.

Further provided is a competitive immunoassay method for detectingquetiapine in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and with quetiapineor a competitive binding partner of quetiapine, wherein one of theantibody and the quetiapine or competitive binding partner thereof islabeled with a detectable marker, and wherein sample quetiapine competeswith the quetiapine or competitive binding partner thereof for bindingto the antibody; and (ii) detecting the label so as to detect samplequetiapine. Further details of the competitive immunoassay method ofdetecting quetiapine in accordance with the subject invention areprovided in the section below entitled “Immunoassays”.

In a preferred embodiment of the subject invention, the detection ofquetiapine is accompanied by the detection of one or more analytes inaddition to quetiapine. Preferably the one or more additional analytesare anti-psychotic drugs other than quetiapine, and more preferably theanti-psychotic drugs other than quetiapine are selected from the groupconsisting of: aripiprazole, risperidone, paliperidone, olanzapine, andmetabolites thereof.

As discussed above, the antibodies of the subject invention can be usedin assays to detect the presence and/or amount of the anti-psychoticdrug in patient samples. Such detection permits therapeutic drugmonitoring enabling all of the benefits thereof. Detection of levels ofanti-psychotic drugs may be useful for many purposes, each of whichrepresents another embodiment of the subject invention, including:determination of patient adherence or compliance with prescribedtherapy; use as a decision tool to determine whether a patient should beconverted from an oral anti-psychotic regimen to a long-actinginjectable anti-psychotic regimen; use as a decision tool to determineif the dose level or dosing interval of oral or injectableanti-psychotics should be increased or decreased to ensure attainment ormaintenance of efficacious or safe drug levels; use as an aid in theinitiation of anti-psychotic drug therapy by providing evidence of theattainment of minimum pK levels; use to determine bioequivalence ofanti-psychotic drug in multiple formulations or from multiple sources;use to assess the impact of polypharmacy and potential drug-druginteractions; and use as an indication that a patient should be excludedfrom or included in a clinical trial and as an aid in the subsequentmonitoring of adherence to clinical trial medication requirements.

Compounds, Conjugates and Immunogens

In relation to the compounds and conjugates and immunogens, thefollowing abbreviations are used: AMAS is N-(α-maleimidoacetoxy)succinimide ester; BINAP is 2,2′-bis(diphenylphosphino)-1,1′-binapthyl;Boc or BOC is tert-butoxycarbonyl; BTG is bovine thyroglobulin; Bu₃N istributylamine; DCC is dicyclohexylcarbodiimide; DCM is dichloromethane;DIEA is diisopropylethylamine; DMF is N,N-dimethylformamide; EDCI or EDCis 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EDTA isethylenediaminetetraaceticacid; HOBT or HOBt is 1-hydroxybenzotriazolehydrate; KLH is keyhole limpet hemocyanin; Pd₂(dba)₃ istris(dibenzylideneacetone) dipalladium(0); SATA is N-succinimidylS-acetylthioacetate; TEA or Et3N is triethylamine; THF istetrahydrofuran; TFA is trifluoroacetic acid; r.t. is room temperature;DEAD is diethylazodicarboxylate; DIC is diisopropylcarbodiimide; NHS isN-hydroxysuccinimide; TFP is Tetrafluorophenyl; PNP is p-nitrophenyl;TBTU is O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate; DEPBT is3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one; BOP-Cl isBis(2-oxo-3-oxazolidinyl)phosphonic chloride; DTT is dithioerythritol.

The term “conjugate” refers to any substance formed from the joiningtogether of separate parts. Representative conjugates include thoseformed by the joining together of a small molecule, such as thecompounds of Formula I, and a large molecule, such as a carrier or apolyamine polymer, particularly a protein. In the conjugate the smallmolecule may be joined at one or more active sites on the largemolecule.

The term “hapten” refers to a partial or incomplete antigen. A hapten isa protein-free substance, which is not capable of stimulating antibodyformation, but which does react with antibodies. The antibodies areformed by coupling a hapten to a high molecular weight immunogeniccarrier, and then injecting this coupled product, i.e., an immunogen,into a human or animal subject.

The term “immunogen” refers to a substance capable of eliciting,producing, or generating an immune response in an organism.

An “immunogenic carrier,” as used herein, is an immunogenic substance,commonly a protein, that can join at one or more positions with haptens,thereby enabling the production of antibodies that can bind with thesehaptens. Examples of immunogenic carrier substances include, but are notlimited to, proteins, glycoproteins, complex polyamino-polysaccharides,particles, and nucleic acids that are recognized as foreign and therebyelicit an immunologic response from the host. Thepolyamino-polysaccharides may be prepared from polysaccharides using anyof the conventional means known for this preparation.

Various protein types may be employed as immunogenic carriers, includingwithout limitation, albumins, serum proteins, lipoproteins, etc.Illustrative proteins include bovine serum albumin, keyhole limpethemocyanin, egg ovalbumin, bovine thyroglobulin, fraction V human serumalbumin, rabbit albumin, pumpkin seed globulin, diphtheria toxoid,tetanus toxoid, botilinus toxin, succinylated proteins, and syntheticpoly(aminoacids) such as polylysine.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymers built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains poly(amino acid) residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “analogue” of a chemical compound refers to a chemical compoundthat contains a chain of carbon atoms and the same particular functionalgroups as a reference compound, but the carbon chain of the analogue islonger or shorter than that of the reference compound.

A “label,” “detector molecule,” “reporter” or “detectable marker” is anymolecule which produces, or can be induced to produce, a detectablesignal. The label can be conjugated to an analyte, immunogen, antibody,or to another molecule such as a receptor or a molecule that can bind toa receptor such as a ligand, particularly a hapten or antibody. A labelcan be attached directly or indirectly by means of a linking or bridgingmoiety. Non-limiting examples of labels include radioactive isotopes(e.g., ¹²⁵I), enzymes (e.g. β-galactosidase, peroxidase), enzymefragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts,fluorophores (e.g., rhodamine, fluorescein isothiocyanate or FITC, orDylight 649), dyes, chemiluminescers and luminescers (e.g., dioxetanes,luciferin), or sensitizers.

As used herein, a “spacer” refers to a portion of a chemical structurewhich connects two or more substructures such as haptens, carriers,immunogens, labels or binding partners through a functional linkinggroup. These spacer groups are composed of the atoms typically presentand assembled in ways typically found in organic compounds and so may bereferred to as “organic spacing groups”. The chemical building blocksused to assemble the spacers will be described hereinafter in thisapplication. Among the preferred spacers are straight or branched,saturated or unsaturated carbon chains. These carbon chains may alsoinclude one or more heteroatoms within the chain, one or moreheteroatoms replacing one or more hydrogens of any carbon atom in thechain, or at the termini of the chains. By “heteroatoms” is meant atomsother than carbon which are chosen from the group consisting of oxygen,nitrogen, phosphorous and sulfur, wherein the nitrogen, phosphorous andsulfur atoms may exist in any oxidation state and may have carbon orother heteroatoms bonded to them. The spacer may also include cyclic oraromatic groups as part of the chain or as a substitution on one of theatoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. Preferred chain lengths are between 1 to 20 atoms.

A “functional linking group” refers to a reactive group that is presenton a hapten and may be used to provide an available reactive sitethrough which the hapten portion may be coupled to another moietythrough formation of a covalent chemical bond to produce a conjugate ofa hapten with another moiety (such as a label or carrier). The haptenmay be linked in this way to a moiety such as biotin to form acompetitive binding partner.

Spacer groups may be used to link the hapten to the carrier. Spacers ofdifferent lengths allow one to attach the hapten with differingdistances from the carrier for presentation to the immune system of theanimal or human being immunized for optimization of the antibodyformation process. Attachment to different positions in the haptenmolecule allows the opportunity to present specific sites on the haptento the immune system to influence antibody recognition. The spacer maycontain hydrophilic solubilizing groups to make the hapten derivativemore soluble in aqueous media. Examples of hydrophilic solubilizinggroups include but are not limited to polyoxyalkyloxy groups, forexample, polyethylene glycol chains; hydroxyl, carboxylate and sulfonategroups.

The term “nucleophilic group” or “nucleophile” refers to a species thatdonates an electron-pair to form a chemical bond in a reaction. The term“electrophilic group” or “electrophile” refers to a species that acceptsan electron-pair from a nucleophile to form a chemical bond in areaction.

The term “substituted” refers to substitution of an atom or group ofatoms in place of a hydrogen atom on a carbon atom in any position onthe parent molecule. Non limiting examples of substituents includehalogen atoms, amino, hydroxy, carboxy, alkyl, aryl, heteroalkyl,heteroaryl, cyano, alkoxy, nitro, aldehyde and ketone groups.

The term “alkyl” refers to saturated or unsaturated linear and branchedchain radicals of up to 12 carbon atoms, unless otherwise indicated, andis specifically intended to include radicals having any degree or levelof saturation. Alkyl includes, but is not limited to, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, hexyl, isohexyl, heptyl, octyl, 2,2,4-trimethylpentyl, nonyl,decyl, undecyl and dodecyl.

The term “cycloalkyl” refers to a saturated or partially unsaturatedmonocyclic or bicyclic hydrocarbon ring radical composed of from 3 to 10carbon atoms. Alkyl substituents may optionally be present on the ring.Examples include cyclopropyl, 1,1-dimethyl cyclobutyl,1,2,3-trimethylcyclopentyl, cyclohexyl and cyclohexenyl.

The term “heteroalkyl” refers to an alkyl group that includes one ormore heteroatoms within the chain, one or more heteroatoms replacing oneor more hydrogens of any carbon atom in the chain, or at termini of thechains.

The term “aminoalkyl” refers to at least one primary or secondary aminogroup bonded to any carbon atom along an alkyl chain.

The term “alkoxy” refers to straight or branched chain radicals of up to12 carbon atoms, unless otherwise indicated, bonded to an oxygen atom.Examples include but are not limited to methoxy, ethoxy, propoxy,isopropoxy and butoxy.

The term “alkoxyalkyl” refers to at least one alkoxy group bonded to anycarbon atom along an alkyl chain.

The term “thioalkyl” refers to at least one sulfur group bonded to anycarbon atom along an alkyl chain. The sulfur group may be at anyoxidation state and includes sulfoxides, sulfones and sulfates.

The term “carboxylate group” includes carboxylic acids and alkyl,cycloalkyl, aryl or aralkyl carboxylate esters.

The term “alkylcarbonyl” refers to a group that has a carbonyl groupbonded to any carbon atom along an alkyl chain.

The term “heteroaryl” refers to 5- to 7-membered mono- or 8- to10-membered bicyclic aromatic ring radicals, any ring of which mayconsist of from one to four heteroatoms selected from N, O or S wherethe nitrogen and sulfur atoms can exist in any allowed oxidation state.Examples include benzimidazolyl, benzothiazolyl, benzothienyl,benzoxazolyl, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl,pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl,thiazolyl and thienyl.

The term “aryl” refers to monocyclic or bicyclic aromatic ring radicalscontaining from 6 to 12 carbons in the ring. Alkyl substituents mayoptionally be present on the ring. Examples include phenyl, biphenyl andnapththalene.

The term “aralkyl” refers to a C₁₋₆ alkyl group containing an arylsubstituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.

The term “acyl” refers to the group —C(O)R_(a), where R_(a) is hydrogen,alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl and heteroaryl. An“acylating agent” adds the —C(O)R_(a) group to a molecule.

The term “sulfonyl” refers to the group —S(O)₂R_(b), where R_(b) ishydrogen, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aryl, aralkyl andheteroaryl. A “sulfonylating agent” adds the —S(O)₂R_(a) group to amolecule.

Spacers bearing reactive functional linking groups for the attachment ofhaptens to carrier moieties may be prepared by a wide variety ofmethods. The spacer may be formed using a molecule that isdifferentially functionalized or activated with groups at either end toallow selective sequential reaction with the hapten and the carrier, butthe same reactive moiety may also be used at both ends. The groupsselected for reaction with the hapten and the functional linking groupto be bound to the carrier are determined by the type of functionalityon the hapten and the carrier that the hapten is to be bonded with.Spacers and methods of attachment to haptens and carriers include butare not limited to those described by Brinkley, M., A., BioconjugateChem. 1992, 3:2-13, Hermanson, Greg T., Bioconjugate Techniques,Academic Press, London, Amsterdam, Burlington, Mass., USA, 2008 andThermo Scientific Pierce Crosslinking Technical Handbook; available fordownload or hard copy request from Thermo Scientific 3747 N Meridian Rd,Rockford, Ill. USA 61101, ph 800-874-3723 or at:http://www.piercenet.com/ and references within. Many differentiallyactivated molecules for formation of spacer groups are commerciallyavailable from vendors, for example Thermo Scientific.

For haptens bearing an amino group, modes of attachment of the spacer tothe hapten include reaction of the amine on the hapten with a spacerbuilding block bearing an acyl halide or active ester. “Active esters”are defined as esters that undergo reaction with a nucleophilic group,for example an amino group, under mild conditions to form a stablelinkage. A stable linkage is defined as one that remains intact underconditions of further use, for example subsequent synthetic steps, useas an immunogen, or in a biochemical assay. A preferred example of astable linkage is an amide bond. Active esters and methods of formationare described by Benoiton, N. L., in Houben-Weyl, Methods of OrganicChemistry, Thieme Stuttgart, New York, vol E22 section 3.2:443 andBenoiton, N. L., Chemistry of Peptide Synthesis, Taylor and Francis, NY, 2006. Preferred active esters include p-nitrophenyl ester (PNP),N-hydroxysuccinimide ester (NHS) and tetrafluorophenyl ester (TFP). Acylhalides may be prepared by many methods known to one skilled in the artfor example, reaction of the carboxylic acid with thionyl chloride oroxalyl chloride, see: Fieser, L. F. and Fieser, M. Reagents for OrganicSynthesis, John Wiley and Sons, N Y, 1967 and references within. Thesemay be converted to other active esters such as p-nitrophenyl esters(PNP) which may also be used in active bi-functional spacers asdescribed by Wu et. al, Organic Letters, 2004, 6 (24):4407.N-hydroxysuccinimide (NHS) esters may be prepared by reaction ofN,N-disuccinimidyl carbonate (CAS 74124-79-1) with the carboxylic acidof a compound in the presence of an organic base such as triethylamineor diisopropylethylamine in an aprotic solvent under anhydrousconditions as described in Example 35 of WO2012012595 or by usingN-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) or otherdehydrating agent, under anhydrous conditions. Tetrafluorophenyl esters(TFP) may be prepared by reaction of carboxylic acids with2,3,5,6-tetrafluorophenyltrifluoroacetate in the presence of an organicbase such as triethylamine or diisopropylethylamine in an aproticsolvent under anhydrous conditions as reported by Wilbur, et. al,Bioconjugate Chem., 2004, 15(1):203. One skilled in the art willrecognize that spacers shown in Table 1, among others, can be obtainedusing known methods and attached to amino-bearing haptens utilizingroutine optimization of reaction conditions. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 1

Reasonable values for m and n are between 1 and 10

Direct coupling of the amine on the hapten and a carboxylic acidfunctionality on the spacer building block in the presence of a couplingagent may also be used as a mode of attachment. Preferred reagents arethose typically used in peptide synthesis. Peptide coupling reagentsinclude but are not limited to0-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU, CAS #125700-67-6), see: Pruhs, S., Org. Process. Res. Dev. 2006,10:441; N-Hydroxybenzotriazole (HOBT, CAS #2592-95-2) with acarbodiimide dehydrating agent, for example N—N-dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), or1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), see:König W., Geiger, R. Chem. Ber., 1970, 103 (3):788;3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one (DEPBT, CAS#165534-43-0), see: Liu, H. et. al., Chinese Chemical Letters, 2002,13(7):601; Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; (BOP-Cl, CAS#68641-49-6), see: Diago-Meseguer, J et. al. Synthesis, 1980, 7:547-51and others described in detail by Benoiton in Chemistry of PeptideSynthesis, CRC Press, Boca Raton, Fla., 2005, Chapter 2, and thetechnical bulletin provided by Advanced Automated Peptide ProteinTechnologies (aapptec), 6309 Shepardsville Rd., Louisville Ky. 40228, ph888 692 9111; www.aapptec.com, and references within. These methodscreate a stable amide linkage attaching the hapten to the spacer.Examples of spacers that can be obtained using known methods andattached to amino-bearing haptens utilizing routine optimization ofreaction conditions employing the methods described and cited above areshown, but not limited to those in Table 2. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 2

reasonable range for n is between 1-10

Spacers may also be constructed in a step-wise fashion by sequentialattachment of appropriate chemical groups to the hapten including thestep of forming the functional linking group that is capable of bindingto the carrier. See illustrative examples under General ReactionSchemes.

Additionally, when the hapten has a nucleophilic group, for example athiol group, an amino group or a hydroxyl group which will become thepoint of attachment of the spacer, the spacer may also be constructed byalkylation of the thiol, amine or hydroxyl group. Any alkyl group thatis appropriately substituted with a moiety capable of undergoing asubstitution reaction, for example, an alkyl halide, or sulfonic acidester such as p-Toluenesulfonate, may be used to attach the spacer. Manyexamples of alkylation reactions are known to one skilled in the art andspecific examples may be found in the general chemical literature andoptimized through routine experimentation. A discussion of alkylationreactions with many references can be found in Chapter 10 of March'sAdvanced Organic Chemistry, Smith, M. B., and March, J., John Wiley &sons, Inc. NY, 2001. Other linkages may also be employed such asreaction of the nucleophilic moiety, for example an amine, on the haptenwith an isocyanate to form a urea or reaction with an isothiocyanate toform a thiourea linkage, see: Li, Z., et. al., Phosphorus, Sulfur andSilicon and the Related Elements, 2003, 178(2):293-297. Spacers may beattached to haptens bearing hydroxyl groups via reaction with isocyanategroups to form carbamate or urethane linkages. The spacer may bedifferentially activated with the isocyanate functional group on one endand a functional linking group capable of reacting with the carrier,see: Annunziato, M. E., Patel, U.S., Ranade, M. and Palumbo, P. S.,Bioconjugate Chem., 1993, 4:212-218.

For haptens bearing a carboxylic acid group, modes of attachment of aspacer portion to the hapten include activation of the carboxylic acidgroup as an acyl halide or active ester, examples of which are shown inTable 3, preparation of which are described previously, followed byreaction with an amino (—NH₂—), hydrazino (—NH—NH₂—), hydrazido(—C(O)—NH—NH₂—) or hydroxyl group (—OH) on the spacer portion to form anamide, hydrazide, diacylhydrazine or ester linkage, or direct couplingof the carboxylic acid group with an amino group on the spacer portionor directly on the carrier with a peptide coupling reagent and/orcarbodiimide dehydrating reagent, described previously, examples ofwhich are shown in Tables 4 and 5. Procedures found in references citedpreviously for formation of activated esters and use of peptide couplingagents may be employed for attachment of carboxylic acid-bearing haptensto spacer building blocks and protein carriers with available aminogroups utilizing routine optimization of reaction conditions.

TABLE 3

Sulfo NHS and NHS

TFP

Acyl chloride

PNP

TABLE 4

HOBT

DEPT

BOP-Cl

TBTU

TABLE 5

diisopropylcarbodiimide (DIC)

Dicyclohexylcarbodiimide (DCC)

1-ethyl-3(3- dimethylaminopropyl)carbodiimide.HCl (EDC)

Other electrophilic groups may be present on the hapten to attach thespacer, for example, a sulfonyl halide

or electrophilic phosphorous group, for example:

See: Malachowski, William P., Coward, James K., Journal of OrganicChemistry, 1994, 59 (25):7616or:

R_(c) is alkyl, cycloalkyl, aryl, substituted aryl, aralkyl.See: Aliouane, L., et. al, Tetrahedron Letters, 2011, 52(28):8681.

Haptens that bear aldehyde or ketone groups may be attached to spacersusing methods including but not limited to reaction with a hydrazidegroup H₂N—NH—C(O)— on the spacer to form an acylhydrazone, see: Chamow,S. M., Kogan, T. P., Peers, D. H., Hastings, R. C., Byrn, R. A. andAskenaszi, A., J. Biol. Chem., 1992, 267(22): 15916. Examples ofbifunctional hydrazide spacer groups that allow attachment to a thiolgroup on the carrier are shown in Table 6.

TABLE 6

Haptens may also contain thiol groups which may be reacted with thecarrier provided that the carrier has been modified to provide a groupthat may react with the thiol. Carrier groups may be modified by methodsincluding but not limited to attachment of a group containing amaleimide functional group by reaction of an amino group on the carrierwith N-Succinimidyl maleimidoacetate, (AMAS, CAS #55750-61-3),Succinimidyl iodoacetate (CAS #151199-81-4), or any of the bifunctionalspacer groups shown in Table 1 to introduce a group which may undergo areaction resulting in attachment of the hapten to the carrier.

The functional linking group capable of forming a bond with the carriermay be any group capable of forming a stable linkage and may be reactiveto a number of different groups on the carrier. The functional linkinggroup may preferably react with an amino group, a carboxylic acid groupor a thiol group on the carrier, or derivative thereof. Non-limitingexamples of the functional linking group are a carboxylic acid group,acyl halide, active ester (as defined previously), isocyanate,isothiocyanate, alkyl halide, amino group, thiol group, maleimide group,acrylate group (H₂C═CH—C(O)—) or vinyl sulfone group H₂C═CH—SO₂—) See:Park, J. W., et. al., Bioconjugate Chem., 2012, 23(3): 350. Thefunctional linking group may be present as part of a differentiallyactivated spacer building block that may be reacted stepwise with thehapten and the resulting hapten derivative may then be reacted with thecarrier. Alternatively, the hapten may be derivatized with a spacer thatbears a precursor group that may be transformed into the functionallinking group by a subsequent reaction. When the functional linkinggroup on the spacer is an amine or a carboxylic acid group, the couplingreaction with the carboxylic acid group or amine on the carrier may becarried out directly through the use of peptide coupling reagentsaccording to procedures in the references cited above for thesereagents.

Particular disulfide groups, for example, pyridyldisulfides, may be usedas the functional linking group on the spacer which may undergo exchangewith a thiol group on the carrier to from a mixed disulfide linkage,see: Ghetie, V., et al., Bioconjugate Chem., 1990, 1:24-31. Thesespacers may be attached by reaction of the amine-bearing hapten with anactive ester which is attached to a spacer bearing the pyridyldisulfidegroup, examples of which include but are not limited to those shown inTable 7.

TABLE 7

Most often the carrier is a protein and the ε-amino groups of the lysineresidues may be used for attachment, either directly by reaction with anamine-reactive functional linking group or after derivitization with athiol-containing group, including N-Succinimidyl S-Acetylthioacetate,(SATA, CAS 76931-93-6), or an analogue thereof, followed by cleavage ofthe actetate group with hydroxylamine to expose the thiol group forreaction with the functional linking group on the hapten. Thiol groupsmay also be introduced into the carrier by reduction of disulfide bondswithin protein carriers with mild reducing reagents including but notlimited to 2-mercaptoethylamine, see: Bilah, M., et. al.,Bioelectrochemistry, 2010, 80(1):49, phosphine reagents, see: Kirley, T.L., Analytical Biochemistry, 1989, 180(2):231 or dithioerythritol (DTT,CAS 3483-12-3) Cleland, W., Biochemistry, 1964, 3:480-482.

General Reaction Schemes

Compounds useful for producing antibodies according to the subjectinvention can be synthesized in accordance with the general syntheticmethods described below. Compounds of Formula (I) can be prepared bymethods known to those who are skilled in the art. The followingreaction schemes are only meant to represent examples of the inventionand are in no way meant to be a limit of the invention.

Derivatives of quetiapine may be prepared by a number of methods. Theprimary hydroxyl group in quetiapine, the starting compound (R₁ andR₂═H) shown in Scheme 1, may be acylated using, for example, succinicanhydride and the method described by Fiedler, H., et. al., Langmuir,1994, 10:3959. The resulting acid may be further functionalized asdescribed elsewhere within this disclosure or attached directly to acarrier using any number of aforementioned methods including those shownin the subsequent examples.

The Primary hydroxyl group of quetiapine may also be alkylated to forman ether according to the procedure of US20100069356, as shown in Scheme2, using an alkyl halide or a sulfonate ester, such as4-bromomethylpentanoate in the presence of tetrabutylamoniumhydrogensulfate and aqueous sodium hydroxide to provide an acid whichmay be used as described above.

Compounds of Formula I where R² is CH₂NHC(O)(CH₂)_(m)CO₂H may be madeaccording to Scheme 3. Reaction of2-(2-(2-(aminomethyl)-4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl)ethoxy)ethanol,prepared as described in Example 1, proceeds with a cyclic anhydridecompound, such as succinic anhydride or glutaric anhydride, in a solventsuch as pyridine, at temperatures ranging from room temperature to 60°C., for about 48 hours. Those skilled in the art will recognize that thesame chemistry may be used to create compounds of Formula I where R¹ isCH₂NHC(O)(CH₂)_(m)CO₂H.

Compounds of Formula I where R² is

may be made according to Scheme 4. Compounds of Formula I, where R² isCH₂NHC(O)(CH₂)_(m)CO₂H, prepared as described in scheme 1, are treatedwith N-t-butoxycarbonylpiperazine, diethyl cyanophosphonate, and a base,such as diisopropylethylamine. The reaction is carried out in a solvent,such as dichloromethane, for about 2 hours at room temperature.Deprotection of the piperazinyl group is accomplished withtrifluoroacetic anhydride as described in Scheme 4, followed by reactionwith an appropriate anhydride, such as succinic anhydride or maleicanhydride, in the presence of a suitable base such asdiisopropylethylamine. Those skilled in the art will recognize that thesame chemistry may be used to create compounds of Formula I where R¹ is

Compounds of Formula I where R¹ is

may be made according to Scheme 5. The maleimide may be introduced byany method known in the art. Maleimide functionalizing groups such as2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetatewhere m is 1, may be used in a solvent such as DMF or CH₂Cl₂, and abase, such as tributylamine or triethylamine. Alternatively, thedeprotected piperazinyl group described in Scheme 4 may be elaboratedwith a maleimide functionality, as described in Scheme 5 to givecompounds of Formula I where R¹ is

Those skilled in the art will recognize that the same chemistry may beused to create compounds of Formula I where R² is

Maleimide functionalized haptens wherein R¹ or R² is

may be conjugated to proteins according to the method shown in Scheme 6.Activation of protein lysine residues by acylation of theepsilon-nitrogen with N-succinimidyl S-acetylthioacetate (SATA),followed by subsequent hydrolysis of the S-acetyl group withhydroxylamine produces a nucleophilic sulfhydryl group. Conjugation ofthe sulfhydryl activated protein with the maleimide derivatized hapten(prepared as described in general scheme 3) proceeds via a Michaeladdition reaction. Suitable proteins are known to those skilled in theart and include keyhole limpet hemocyanin, bovine thyroglobulin, andovalbumin. The same methodology may be used to conjugate proteins tomaleimide functionalized haptens where R¹ or R² is

Carboxylic acid functionalized haptens, wherein R¹ or R² isCH₂NHC(O)(CH₂)_(m)CO₂H, may be conjugated to proteins according to themethod shown in Scheme 7. Reaction with N-hydroxysuccinimide and asuitable coupling agent, such as dicyclohexylcarbodiimide, and a base,such as tributyl amine, in a solvent such as DMF, at a temperature ofabout 20° C., for about 18 hrs activates the carboxylic acid with thehydroxypyrrolidine-2,5-dione leaving group. The activated linker andhapten may then be conjugated to a protein in a solvent, such as a pH7.5 phosphate buffer, at about 20° C., for about 2.5 hours. Suitableproteins are known to those skilled in the art and include keyholelimpet hemocyanin, bovine thyroglobulin, and ovalbumin. The samemethodology may be used to conjugate proteins to carboxylic acidfunctionalized haptens where R¹ or R² is

Antibodies

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to quetiapine and which: (i) is generatedin response to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i). The term “antibody” refers to a specificprotein capable of binding an antigen or portion thereof (in accordancewith this invention, capable of binding to an anti-psychotic drug ormetabolite thereof). An antibody is produced in response to an immunogenwhich may have been introduced into a host, e.g., an animal or a human,by injection. The generic term “antibody” includes polyclonalantibodies, monoclonal antibodies, and antibody fragments.

“Antibody” or “antigen-binding antibody fragment” refers to an intactantibody, or a fragment thereof, that competes with the intact antibodyfor binding. Generally speaking, an antibody or antigen-binding antibodyfragment, is said to specifically bind an antigen when the dissociationconstant is less than or equal to 1 μM, preferably less than or equal to100 nM and most preferably less than or equal to 10 nM. Binding can bemeasured by methods know to those skilled in the art, an example beingthe use of a BIAcore™ instrument.

Antibody fragments comprise a portion of an intact antibody, preferablythe antigen binding or variable region of the intact antibody. Bindingfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. An antibody other than a“bispecific” or “bifunctional” antibody is understood to have each ofits binding sites identical.

As used herein, “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Two antibodies are said to “bind thesame epitope” if one antibody is shown to compete with the secondantibody in a competitive binding assay, by any of the methods wellknown to those skilled in the art (such as the BIAcore™ method referredto above). In reference to a hapten (such as quetiapine or otheranti-psychotic drug), an antibody can be generated against thenon-antigenic hapten molecule by conjugating the hapten to animmunogenic carrier. An antibody is then generated which recognizes an“epitope” defined by the hapten.

“Isolated” when used in the context of an antibody means altered “by thehand of man” from any natural state; i.e., that, if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a naturally occurring antibody naturally present in aliving animal in its natural state is not “isolated”, but the sameantibody separated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. Antibodies may occur in acomposition, such as an immunoassay reagent, which are not naturallyoccurring compositions, and therein remain isolated antibodies withinthe meaning of that term as it is employed herein.

“Cross-reactivity” refers to the reaction of an antibody with an antigenthat was not used to induce that antibody.

Preferably, the antibody of the subject invention will bind to the drugand any desired pharmacologically active metabolites. By altering thelocation of the attachment of the immunogenic carrier to the compoundsof the invention, selectivity and cross-reactivity with metabolites canbe engineered into the antibodies. For quetiapine, cross-reactivity withquetiapine metabolites such as N-desalkylquetiapine (norquetiapine),quatiapine sulfoxide, 0-desalkylquetiapine or 7-hydroxy quetiapine mayor may not be desirable. Antibodies may be generated that detectmultiple ones of these drugs and/or metabolites, or antibodies may begenerated that detect each separately (thus defining the antibody“specific binding” properties). An antibody specifically binds one ormore compounds when its binding of the one or more compounds isequimolar or substantially equimolar.

Methods of producing such antibodies comprise inoculating a host withthe conjugate described herein. Suitable hosts include, but are notlimited to, mice, rats, hamsters, guinea pigs, rabbits, chickens,donkeys, horses, monkeys, chimpanzees, orangutans, gorillas, humans, andany species capable of mounting a mature immune response. Theimmunization procedures are well established in the art and are setforth in numerous treatises and publications including “The ImmunoassayHandbook”, 2nd Edition, edited by David Wild (Nature Publishing Group,2000) and the references cited therein.

Preferably, an immunogen embodying features of the present invention isadministered to a host subject, e.g., an animal or human, in combinationwith an adjuvant. Suitable adjuvants include, but are not limited to,Freund's adjuvant, powdered aluminum hydroxide (alum), aluminumhydroxide together with Bordetella pertussis, and monophosphoryl lipidA-synthetic trehalose dicorynomycolate (MPL-TDM).

Typically, an immunogen or a combination of an immunogen and an adjuvantis injected into a mammalian host by one or multiple subcutaneous orintraperitoneal injections. Preferably, the immunization program iscarried out over at least one week, and more preferably, over two ormore weeks. Polyclonal antibodies produced in this manner can beisolated and purified utilizing methods well know in the art.

Monoclonal antibodies can be produced by the well-established hybridomamethods of Kohler and Milstein, e.g., Nature 256:495-497 (1975).Hybridoma methods typically involve immunizing a host or lymphocytesfrom a host, harvesting the monoclonal antibody secreting or having thepotential to secrete lymphocytes, fusing the lymphocytes to immortalizedcells, and selecting cells that secrete the desired monoclonal antibody.

A host can be immunized to elicit lymphocytes that produce or arecapable of producing antibodies specific for an immunogen.Alternatively, the lymphocytes can be immunized in vitro. If human cellsare desired, peripheral blood lymphocytes can be used, although spleencells or lymphocytes from other mammalian sources are preferred.

The lymphocytes can be fused with an immortalized cell line to formhybridoma cells, a process which can be facilitated by the use of afusing agent, e.g., polyethylene glycol. By way of illustration, mutantrodent, bovine, or human myeloma cells immortalized by transformationcan be used. Substantially pure populations of hybridoma cells, asopposed to unfused immortalized cells, are preferred. Thus, followingfusion, the cells can be grown in a suitable medium that inhibits thegrowth or survival of unfused, immortalized cells, for example, by usingmutant myeloma cells that lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT). In such an instance, hypoxanthine,aminopterin, and thymidine can be added to the medium (HAT medium) toprevent the growth of HGPRT-deficient cells while permitting hybridomasto grow.

Preferably, immortalized cells fuse efficiently, can be isolated frommixed populations by selection in a medium such as HAT, and supportstable and high-level expression of antibody following fusion. Preferredimmortalized cell lines include myeloma cell lines available from theAmerican Type Culture Collection, Manassas, Va.

Because hybridoma cells typically secrete antibody extracellularly, theculture media can be assayed for the presence of monoclonal antibodiesspecific for the anti-psychotic drug. Immunoprecipitation of in vitrobinding assays, for example, radiioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA), can be used to measure the bindingspecificity of monoclonal antibodies.

Monoclonal antibody-secreting hybridoma cells can be isolated as singleclones by limiting dilution procedures and sub-cultured. Suitableculture media include, but are not limited to, Dulbecco's ModifiedEagle's Medium, RPMI-1640, and polypeptide-free, polypeptide-reduced, orserum-free media, e.g., Ultra DOMA PF or HL-1, available fromBiowhittaker, Walkersville, Md. Alternatively, the hybridoma cells canbe grown in vivo as ascites.

Monoclonal antibodies can be isolated and/or purified from a culturemedium or ascites fluid by conventional immunoglobulin (Ig) purificationprocedures including, but not limited to, polypeptide A-SEPHAROSE,hydroxylapatite chromatography, gel electrophoresis, dialysis, ammoniumsulfate precipitation, and affinity chromatography.

Monoclonal antibodies can also be produced by recombinant methods suchas are described in U.S. Pat. No. 4,166,452. DNA encoding monoclonalantibodies can be isolated and sequenced using conventional procedures,e.g., using oligonucleotide probes that specifically bind to murineheavy and light antibody chain genes, preferably to probe DNA isolatedfrom monoclonal antibody hybridoma cells lines secreting antibodiesspecific for anti-psychotic drugs.

Antibody fragments which contain specific binding sites for theanti-psychotic drug may also be generated. Such fragments include, butare not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al., Science 256:1270-1281 (1989)). Fab, Fvand ScFv antibody fragments can all be expressed in and secreted fromEscherichia coli, allowing for the production of large amounts of thesefragments. Alternatively, Fab′-SH fragments can be directly recoveredfrom E. coli and chemically coupled to form F(ab′)₂ fragments (Carter etal., BioTechnology 10:163-167 (1992)). Other techniques for theproduction of antibody fragments are known to those skilled in the art.Single chain Fv fragments (scFv) are also envisioned (see U.S. Pat. Nos.5,761,894 and 5,587,458). Fv and sFv fragments are the only species withintact combining sites that are devoid of constant regions; thus, theyare likely to show reduced non-specific binding. The antibody fragmentmay also be a “linear antibody” e.g., as described in U.S. Pat. No.5,642,870, for example. Such linear antibody fragments may bemonospecific or bispecific.

Assay Kits and Devices

An assay kit (also referred to as a reagent kit) can also be providedcomprising an antibody as described above. A representative reagent kitmay comprise an antibody that binds to the anti-psychotic drug,quetiapine, a complex comprising an analog of an anti-psychotic drug ora derivative thereof coupled to a labeling moiety, and may optionallyalso comprise one or more calibrators comprising a known amount of ananti-psychotic drug or a related standard.

The phrase “assay kit” refers to an assembly of materials and reagentsthat is used in performing an assay. The reagents can be provided inpackaged combination in the same or in separate containers, depending ontheir cross-reactivities and stabilities, and in liquid or inlyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. An assay kit embodying features of the presentinvention comprises antibodies which bind quetiapine. The kit mayfurther comprise competitive binding partners of quetiapine andcalibration and control materials.

The phrase “calibration and control material” refers to any standard orreference material containing a known amount of an analyte. A samplesuspected of containing an analyte and the corresponding calibrationmaterial are assayed under similar conditions. The concentration ofanalyte is calculated by comparing the results obtained for the unknownspecimen with the results obtained for the standard. This is commonlydone by constructing a calibration curve.

Antibodies embodying features of the present invention can be includedin a kit, container, pack, or dispenser together with instructions fortheir utilization. When the antibodies are supplied in a kit, thedifferent components of the immunoassay may be packaged in separatecontainers and admixed prior to use. Such packaging of the componentsseparately may permit long-term storage without substantiallydiminishing the functioning of the active components. Furthermore,reagents can be packaged under inert environments, e.g., under apositive pressure of nitrogen gas, argon gas, or the like, which isespecially preferred for reagents that are sensitive to air and/ormoisture.

Reagents included in kits embodying features of the present inventioncan be supplied in all manner of containers such that the activities ofthe different components are substantially preserved while thecomponents themselves are not substantially adsorbed or altered by thematerials of the container. Suitable containers include, but are notlimited to, ampules, bottles, test tubes, vials, flasks, syringes,envelopes, e.g., foil-lined, and the like. The containers may becomprised of any suitable material including, but not limited to, glass,organic polymers, e.g., polycarbonate, polystyrene, polyethylene, etc.,ceramic, metal, e.g., aluminum, metal alloys, e.g., steel, cork, and thelike. In addition, the containers may comprise one or more sterileaccess ports, e.g., for access via a needle, such as may be provided bya septum. Preferred materials for septa include rubber andpolytetrafluoroethylene of the type sold under the trade name TEFLON byDuPont (Wilmington, Del.). In addition, the containers may comprise twoor more compartments separated by partitions or membranes that can beremoved to allow mixing of the components.

Reagent kits embodying features of the present invention may also besupplied with instructional materials. Instructions may be printed,e.g., on paper and/or supplied in an electronically-readable medium.Alternatively, instructions may be provided by directing a user to aninternet website, e.g., specified by the manufacturer or distributor ofthe kit and/or via electronic mail.

The antibody may also be provided as part of an assay device. Such assaydevices include lateral flow assay devices. A common type of disposablelateral flow assay device includes a zone or area for receiving theliquid sample, a conjugate zone, and a reaction zone. These assaydevices are commonly known as lateral flow test strips. They employ aporous material, e.g., nitrocellulose, defining a path for fluid flowcapable of supporting capillary flow. Examples include those shown inU.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 all ofwhich are incorporated herein by reference in their entireties.

Another type of assay device is a non-porous assay device havingprojections to induce capillary flow. Examples of such assay devicesinclude the open lateral flow device as disclosed in PCT InternationalPublication Nos. WO 2003/103835, WO 2005/089082, WO 2005/118139, and WO2006/137785, all of which are incorporated herein by reference in theirentireties.

In a non-porous assay device, the assay device generally has at leastone sample addition zone, at least one conjugate zone, at least onereaction zone, and at least one wicking zone. The zones form a flow pathby which sample flows from the sample addition zone to the wicking zone.Also included are capture elements, such as antibodies, in the reactionzone, capable of binding to the analyte, optionally deposited on thedevice (such as by coating); and a labeled conjugate material alsocapable of participating in reactions that will enable determination ofthe concentration of the analyte, deposited on the device in theconjugate zone, wherein the labeled conjugate material carries a labelfor detection in the reaction zone. The conjugate material is dissolvedas the sample flows through the conjugate zone forming a conjugate plumeof dissolved labeled conjugate material and sample that flows downstreamto the reaction zone. As the conjugate plume flows into the reactionzone, the conjugated material will be captured by the capture elementssuch as via a complex of conjugated material and analyte (as in a“sandwich” assay) or directly (as in a “competitive” assay). Unbounddissolved conjugate material will be swept past the reaction zone intothe at least one wicking zone. Such devices can include projections ormicropillars in the flow path.

An instrument such as that disclosed in US Patent Publication Nos.US20060289787A1 and US 20070231883A1, and U.S. Pat. Nos. 7,416,700 and6,139,800, all of which are incorporated herein by reference in theirentireties, is able to detect the bound conjugated material in thereaction zone. Common labels include fluorescent dyes that can bedetected by instruments which excite the fluorescent dyes andincorporate a detector capable of detecting the fluorescent dyes.

Immunoassays

The antibodies thus produced can be used in immunoassays torecognize/bind to the anti-psychotic drug, thereby detecting thepresence and/or amount of the drug in a patient sample. Preferably, theassay format is a competitive immunoassay format. Such an assay formatand other assays are described, among other places, in Hampton et al.(Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

The term “analyte” refers to any substance or group of substances, thepresence or amount of which is to be determined. Representativeanti-psychotic drug analytes include, but are not limited to,risperidone, paliperidone, olanzapine, aripiprazole, and quetiapine.

The term “competitive binding partner” refers to a substance or group ofsubstances, such as may be employed in a competitive immunoassay, whichbehave similarly to an analyte with respect to binding affinity to anantibody. Representative competitive binding partners include, but arenot limited to, anti-psychotic drug derivatives and the like.

The term “detecting” when used with an analyte refers to anyquantitative, semi-quantitative, or qualitative method as well as to allother methods for determining an analyte in general, and ananti-psychotic drug in particular. For example, a method that merelydetects the presence or absence of an anti-psychotic drug in a samplelies within the scope of the present invention, as do methods thatprovide data as to the amount or concentration of the anti-psychoticdrug in the sample. The terms “detecting”, “determining”, “identifying”,and the like are used synonymously herein, and all lie within the scopeof the present invention.

A preferred embodiment of the subject invention is a competitiveimmunoassay wherein antibodies which bind the anti-psychotic drug, orthe drug or competitive binding partner thereof, are attached to a solidsupport (such as the reaction zone in a lateral flow assay device) andlabeled drug or competitive binding partner thereof, or labeledantibody, respectively, and a sample derived from the host are passedover the solid support and the amount of label detected attached to thesolid support can be correlated to a quantity of drug in the sample.

Any sample that is suspected of containing an analyte, e.g., ananti-psychotic drug, can be analyzed in accordance with the methods ofthe presently preferred embodiments. The sample can be pretreated ifdesired and can be prepared in any convenient medium that does notinterfere with the assay. Preferably, the sample comprises an aqueousmedium such as a body fluid from a host, most preferably plasma orserum.

It is to be understood that all manner of immunoassays employingantibodies are contemplated for use in accordance with the presentlypreferred embodiments, including assays in which antibodies are bound tosolid phases and assays in which antibodies are in liquid media. Methodsof immunoassays that can be used to detect analytes using antibodiesembodying features of the present invention include, but are not limitedto, competitive (reagent limited) assays wherein labeled analyte(analyte analog) and analyte in a sample compete for antibodies andsingle-site immunometric assays wherein the antibody is labeled; and thelike.

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Habor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Copending applications entitled “Haptens of Aripiprazole”, U.S.Provisional Patent Appl. No. 61/691,450, filed Aug. 21, 2012, and US20140163206, filed Aug. 20, 2013), “Haptens of Olanzapine”, (U.S.Provisional Patent Appl. No. 61/691,454, filed Aug. 21, 2012, and US20140213766, filed Aug. 20, 2013), “Haptens of Paliperidone”, U.S.Provisional Patent Appl. No. 61/691,459, filed Aug. 21, 2012, and US20140213767, filed Aug. 20, 2013), “Haptens of Quetiapine” (U.S.Provisional Patent Appl. No. 61/691,462, filed Aug. 21, 2012, and US20140221616, filed Aug. 20, 2013), “Haptens of Risperidone andPaliperidone” (U.S. Provisional Patent Appl. No. 61/691,469, filed Aug.21, 2012, and US 20140155585, Aug. 20, 2013), “Antibodies toAripiprazole Haptens and Use Thereof” (U.S. Provisional Patent Appl. No.61/691,544, filed Aug. 21, 2012, and US 20140057299, filed Aug. 20,2013), “Antibodies to Olanzapine Haptens and Use Thereof” (U.S.Provisional Patent Appl. No. 61/691,572, filed Aug. 21, 2012, US20140057303, filed Aug. 20, 2013), “Antibodies to Paliperidone Haptensand Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,634, filedAug. 21, 2012, and US 20140057297, filed Aug. 20, 2013), “Antibodies toRisperidone Haptens and Use Thereof” (U.S. Provisional Patent Appl. No.61/691,615, filed Aug. 21, 2012, and US 20140057301, filed Aug. 20,2013), “Antibodies to Aripiprazole and Use Thereof” (U.S. ProvisionalPatent Appl. No. 61/691,522, filed Aug. 21, 2012, and US 20140057300,filed Aug. 20, 2013), “Antibodies to Olanzapine and Use Thereof” (U.S.Provisional Patent Appl. No. 61/691,645, filed Aug. 21, 2012, and US20140057304, filed Aug. 20, 2013), “Antibodies to Paliperidone and UseThereof” (U.S. Provisional Patent Appl. No. 61/691,692, filed Aug. 21,2012, and US 20140057298, filed Aug. 20, 2013), “Antibodies toQuetiapine and Use Thereof” (U.S. Provisional Patent Appl. No.61/691,659, filed Aug. 21, 2012, and US 20140057306, filed Aug. 20,2013), “Antibodies to Risperidone and Use Thereof” (U.S. ProvisionalPatent Appl. No. 61/691,675, filed Aug. 21, 2012, and US 20140057302,filed Aug. 20, 2013), and “Antibodies to Risperidone and Use Thereof”(U.S. Provisional Patent Appl. No. 61/790,880, filed Mar. 15, 2013, andUS 20140057302, filed Aug. 20, 2013) are all incorporated herein byreference in their entireties.

Example 12-(2-(2-(aminomethyl)-4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl)ethoxy)ethanolStep A Piperazine-2-carbonitrile

A stirred solution of tetrahydrofuran (300 mL) and ethylenediamine(108.2 g) at 30° C. was treated dropwise with 2-chloroacrylonitrile(105.0 g) over a period of 2 hours and stirred for 6 additional hours at30° C. The reaction mixture was cooled to 20° C. and a precipitateformed. The reaction was filtered, and the filtrate was adjust pH to 4by adding 35% hydrochloric acid. The resulting precipitate was collectedby filtration. The combined precipitates were dissolved in 20%hydrochloric acid solution and then poured into THF solution toprecipitate the title compound, which was dried under reduced pressureand used in the next reaction without additional purification. ¹H NMR:(D₂O, 400 MHz): δ (ppm) 5.00-4.97 (m, 1H), 3.79 (d, J=4.8 Hz, 2H),3.62-3.44 (m, 4H).

Step B tert-Butyl 3-cyanopiperazine-1-carboxylate

To a solution of compound piperazine-2-carbonitrile, prepared asdescribed in the previous step, (90.6 g, 0.492 mol) was addedtriethylamine (206 mL, 1.476 mol) and Boc₂O (117 g, 0.542 mol). Thereaction mixture was stirred at room temperature overnight, and thenconcentrated. The residue was purified by silica gel chromatography toprovide the title compound.

¹H NMR: (CDCl₃, 400 MHz): δ (ppm) 4.06-3.91 (m, 3H), 3.28-2.83 (m, 4H),1.47 (s, 9H).

Step C tert-Butyl3-cyano-4-(2-(2-hydroxyethoxy)ethyl)piperazine-1-carboxylate

A solution of tert-butyl 3-cyanopiperazine-1-carboxylate, prepared asdescribed in the previous step, (10 g, 0.047 mol) and2-(2-hydroxyethoxy)acetaldehyde (14.8 g) (see: Bodin, A., ContactDermatitis, 2001, 44:207) in dichloromethane was treated with formicacid (12.7 g), and the reaction mixture was stirred at room temperatureovernight. Sodium cyanoborohydride (7.2 g, 0.118 mol) was added inportions. The reaction mixture was stirred at room temperature for 3hours followed by the addition of water and extraction withdichloromethane. The organic layer was washed with brine, dried oversodium sulfate, filtered, and concentrated. The crude product waspurified by column chromatography to provide the product.

¹H NMR: (CDCl₃, 400 MHz): δ (ppm) 4.15 (s, 1H), 3.69-3.63 (m, 4H), 3.58(d, J=4.4 Hz, 2H), 3.47-3.44 (m, 4H), 2.61 (d, J=5.2 Hz, 2H), 2.51-2.48(m, 4H), 1.43 (s, 9H).

Step D tert-Butyl3-(aminomethyl)-4-(2-(2-hydroxyethoxy)ethyl)piperazine-1-carboxylate

To a solution of tert-butyl3-cyano-4-(2-(2-hydroxyethoxy)ethyl)piperazine-1-carboxylate, preparedas described in the previous step, (9.9 g, 33.1 mmol) in methanol (20mL) was added Raney Ni (15 g). The reaction solution was stirred at roomtemperature overnight under hydrogen atmosphere (50 psi). The mixturewas filtered and concentrated to provide the product, which was used inthe next step without additional purification.

ESI-MS (M+1): 304 calc. for C₁₄H₂₉N₃O₄ 303.

Step E tert-Butyl4-(2-(2-hydroxyethoxy)ethyl)-3-((2,2,2-trifluoroacetamido)methyl)piperazine-1-carboxylate

To a solution of tert-butyl3-(aminomethyl)-4-(2-(2-hydroxyethoxy)ethyl)piperazine-1-carboxylate,prepared as described in the previous step (8.8 g) in dichloromethane(100 mL) was added triethylamine (8.8 g, 87.0 mmol) and trifluoroaceticanhydride (6.1 g, 29.0 mmol). The reaction mixture was stirred at roomtemperature for 12 h, diluted with dichloromethane and washed withwater. The organic layer was washed with brine, dried over sodiumsulfate, filtered, and concentrated to give the crude product which waspurified by column chromatography to provide the title compound.

ESI-MS (M+1): 400 calc. for C₁₆H₂₈F₃N₃O₅ 399.

Step F2,2,2-Trifluoro-N-((1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)acetamide

A solution of tert-butyl4-(2-(2-hydroxyethoxy)ethyl)-3-((2,2,2-trifluoroacetamido)methyl)piperazine-1-carboxylate,prepared as described in the previous step, (8.6 g, crude) in methanolichydrogen chloride (20 mL) was stirred at room temperature for 1 hour,followed by concentration to provide the title compound which was usedwithout further purification.

ESI-MS (M+1): 300 calc. for C₁₁H₂₀F₃N₃O₃ 299.

Step G 2-((2-Nitrophenyl)thio)benzoic acid

To a solution of 2-mercapto-benzoic acid (30 g, 0.195 mol) inisopropanol (500 mL) at room temperature were added1-fluoro-2-nitro-benzene (30.2 g, 0.214 mol), water (100 mL) andpotassium hydroxide (31.1 g, 0.555 mol). The reaction mixture wasstirred at room temperature overnight, quenched with water and dilutedwith ethyl acetate. The aqueous phase was extracted with ethyl acetate(3×400 mL) and the combined organic extracts were washed with saturatedaqueous sodium chloride (500 mL), dried over magnesium sulfate,filtered, and concentrated. The crude residue was purified by flashcolumn chromatography on silica gel to give the title compound. ESI-MS(M+1): 276 calc. for C₁₃H₉NO₄S 275. ¹H NMR: (CDCl₃, 400 MHz): δ (ppm)8.12-8.07 (m, 2H), 7.54-7.43 (m, 2H), 7.42-7.39 (m, 2H), 7.35-7.31 (m,1H), 7.12-7.09 (m, 1H).

Step H 2-((2-Aminophenyl)thio)benzoic acid

To a solution of 2-((2-nitrophenyl)thio)benzoic acid, prepared asdescribed in the previous step, (43.3 g, 0.157 mol) in ethyl acetate(500 mL) was added Pd/C (8 g). The reaction solution was stirred at roomtemperature overnight under hydrogen gas atmosphere. The mixture wasfiltered and concentrated to provide the title compound. ESI-MS (M+1):246 calc. for C₁₃H₁₁NO₂S 245. ¹H NMR: (CDCl₃, 400 MHz): δ (ppm)8.20-8.17 (m, 1H), 7.51-7.48 (m, 1H), 7.36-7.30 (m, 2H), 7.21-7.17 (m,1H), 6.88-6.80 (m, 3H).

Step I Dibenzo[b,f][1,4]thiazepin-11(10H)-one

To a solution of 2-((2-aminophenyl)thio)benzoic acid, prepared asdescribed in the previous step, (30 g, 0.122 mol) in dichloromethane(300 mL) was added EDCI (35.2 g, 0.183 mol), triethylamine (51 mL, 0.366mol) and HOBT (24.7 g, 0.183 mol). The reaction mixture was stirred atroom temperature for 12 hours, washed with 1M aq.HCl, saturated aqueoussodium bicarbonate, saturated aqueous sodium chloride, and dried overMgSO₄. The solution was filtered, concentrated, and purified by columnchromatography to provide the title compound. ESI-MS (M+1): 228 calc.for C₁₃H₉NOS 227. ¹H NMR: (CDCl₃, 400 MHz): δ (ppm) 7.70-7.67 (m, 1H),7.58-7.52 (m, 2H), 7.50-7.42 (m, 2H), 7.39-7.35 (m, 1H), 7.24-7.22 (m,1H), 7.17-7.13 (m, 1H).

Step J 11-Chlorodibenzo[b,f][1,4]thiazepine

A solution of dibenzo[b,f][1,4]thiazepin-11(10H)-one, prepared asdescribed in the previous step, (14.6 g, 64 mmol) in phosphorusoxychloride (20 mL) was heated to reflux for 2 hours. The mixture wasconcentrated to provide the crude product which was used directlywithout further purification. ESI-MS (M+1): 246 calc. for C₁₃H₈ClNS 245.

Step KN-((4-(Dibenzo[b,f][1,4]thiazepin-11-yl)-1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)-2,2,2-trifluoroacetamide

To a solution of 11-chlorodibenzo[b,f][1,4]thiazepine, prepared asdescribed in the previous step, (2 g, crude) in dioxane (20 mL) wasadded Pd₂(dba)₃ (327 mg, 0.357 mmol), BINAP (225 mg, 0.357 mmol),triethylamine (6 mL, 42.9 mmol) and2,2,2-trifluoro-N-((1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)acetamide,prepared as described as Step F, (2.4 g, crude). The resulting mixturewas heated to reflux overnight under nitrogen atmosphere, filteredthrough CELITE™, and concentrated. The residue was purified by silicagel chromatography to provide the title compound. ESI-MS (M+1): 509calc. for C₂₄H₂₇F₃N₄O₃S 508.

Step L2-(2-(2-(Aminomethyl)-4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl)ethoxy)ethanol

A mixture ofN-((4-(dibenzo[b,f][1,4]thiazepin-11-yl)-1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)-2,2,2-trifluoroacetamide,prepared as described in the previous step, (2.0 g) and aqueouspotassium carbonate (5%) (15 mL) in methanol (20 mL) was stirred at roomtemperature for 18 hours and extracted with ethyl acetate. The organiclayers were washed with saturated aqueous sodium chloride, dried oversodium sulfate, filtered, evaporated to give the crude product which waspurified by column chromatography, and followed by prep-HPLC to providethe title compound as a yellow solid. ESI-MS (M+1): 413 calc. forC₂₂H₂₈N₄O₂S 412. ¹H NMR: (CDCl₃, 400 MHz): δ (ppm) 7.52-7.50 (m, 1H),7.41-7.31 (m, 4H), 7.17-7.12 (m, 1H), 7.02-7.00 (m, 1H), 6.89-6.84 (m,1H), 3.66-3.59 (m, 5H), 3.54-3.51 (m, 2H), 3.49-3.38 (m, 1H), 3.19-3.12(m, 1H), 3.03-2.88 (m, 2H), 2.79-2.53 (m, 5H).

Example 2N-((4-(Dibenzo[b,f][1,4]thiazepin-11-yl)-1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide

To a solution of2-(2-(2-(aminomethyl)-4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl)ethoxy)ethanol,prepared as described in Example 1, (7.8 mg, 19.0 μmoles) in 410 μL ofDMF and 8.9 μL of tributylamine was added 480 μL of a DMF solution ofN-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 4.8 mg, 19.0μmoles). The resulting solution was allowed to stir for 60 minutes at20° C., then used as such in conjugation reaction with thiol-activatedprotein.

Example 32-{2-[4-(3-Aminomethyl-dibenzo[b,f][1,4]thiazepin-11-yl)-piperazin-1-yl]-ethoxy}-ethanol

Step A 11-Oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-3-carboxylic acid

A mixture of 2-amino-benzenethiol (1.34 mL, 12.5 mmol),2-bromo-terephthalic acid (1.54 g, 6.3 mmol), cuprous oxide (0.50 g, 3.5mmol), quinoline (6.3 mL), and pyridine (0.63 mL) was heated in a 180°C. oil bath under nitrogen for 20 hours, then cooled to roomtemperature. Concentrated hydrochloric acid (20 mL) was added slowlywhile cooling in cold water, with stirring. The resulting precipitatewas filtered, washed with water, and dried to give crude title compound(2 g). LC-MS: m/z 270 (M-1).

Step B 11-Chloro-dibenzo[b,f][1,4]thiazepine-3-carbonyl chloride

To a suspension of11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-3-carboxylic acid,prepared as described in the previous step, (0.41 g) in toluene (6.5 mL)was added DMF (0.125 mL) and thionyl chloride (6.5 mL). The mixture washeated in an 80° C. oil bath under nitrogen over night. The resultingsolution was concentrated to dryness. The crude product was used fornext step.

Step C 11-Chloro-dibenzo[b,f][1,4]thiazepine-3-carboxylic acid amide

A solution of 11-chloro-dibenzo[b,f][1,4]thiazepine-3-carbonyl chloride,prepared as described in the previous step, (ca 1.5 mmol) indichloromethane (10 mL) was treated with a 1,4-dioxane solution ofammonia (0.5 M, 9 mL) under ice bath. The resulting suspension wasstirred at room temperature for 1 hour, and the reaction was quenchedwith water (10 mL). The resulting precipitate was filtered, washed withwater and dichloromethane, and dried. The organic layer of the filtratewas washed with saturated aqueous sodium bicarbonate solution andconcentrated to additional off white product, which was used in the nextstep without additional purification. LC-MS: m/z 289 (M+1). ¹H NMR(DMSO-d₆, 400 MHz): δ (ppm) 8.19 (br, 1H), 8.00-7.96 (m, 2H), 7.90 (d,1H), 7.64 (br, 1H), 7.56 (m, 1H), 7.47 (m, 1H), 7.31 (m, 2H).

Step D11-{4-[2-(2-Hydroxy-ethoxy)-ethyl]-piperazin-1-yl}-dibenzo[b,f][1,4]thiazepine-3-carboxylicacid amide

To a solution of 11-chloro-dibenzo[b,f][1,4]thiazepine-3-carboxylic acidamide, prepared as described in the previous step, (0.40 g) in DMF (1.5mL) and toluene (1.5 mL) was added 2-(2-piperazin-1-yl-ethoxy)-ethanol(0.50 g, 2.9 mmol). The solution was heated in a 110° C. oil bath undernitrogen for 5 hours, concentrated, and purified (silica gel, 2-5%methanol-dichloromethane containing ammonia eluent) to give the titlecompound as an off white solid. LC-MS: m/z 427 (M+1).

Step E2-{2-[4-(3-Aminomethyl-dibenzo[b,f][1,4]thiazepin-11-yl)-piperazin-1-yl]-ethoxy}-ethanol

To a solution of2-{2-[4-(3-aminomethyl-dibenzo[b,f][1,4]thiazepin-11-yl)-piperazin-1-yl]-ethoxy}-ethanol,prepared as described in the previous step, (0.24 g, 0.56 mmol) in THF(15 mL) was added 1 M lithium aluminum hydride THF solution (6 mL, 6mmol). The white suspension was heated in a 70° C. oil bath undernitrogen for 2 hours. The reaction suspension was quenched with slowaddition of saturated aqueous sodium sulfate solution under ice bath.The solution phase was separated, and solid was extracted with THF (5×10mL). The combined organic phases were concentrated and purified (silicagel, 2-5% methanol-dichloromethane containing ammonia eluent) to givethe title compound as an off white solid. LC-MS: m/z 413 (M+1). ¹H NMR(CDCl₃, 400 MHz) δ (ppm) 7.47 (s, 1H), 7.38 (m, 1H), 7.26 (m, 2H,overlapped with solvent), 7.17 (m, 1H), 7.06 (m, 1H), 6.88 (m, 1H), 3.85(s, 2H), 3.76-3.46 (m, 11H, containing exchangeable protons), 2.66-2.57(m, 8H).

Example 42-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((11-(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)dibenzo[b,f][1,4]thiazepin-3-yl)methyl)acetamide

To a solution of2-{2-[4-(3-aminomethyl-dibenzo[b,f][1,4]thiazepin-11-yl)-piperazin-1-yl]-ethoxy}-ethanol,prepared as described in Example 3, (5.6 mg, 13.6 μmoles) in 295 μL ofDMF and 6.4 μL of tributylamine was added 340 μL of a DMF solution ofN-(α-maleimidoacetoxy)succinimide ester (AMAS, 10 mg/mL, 3.4 mg, 13.6μmoles). The resulting solution was allowed to stir for 60 minutes at20° C., then used as such in conjugation reaction with thiol-activatedprotein.

Example 5N-((4-(dibenzo[b,f][1,4]thiazepin-11-yl)-1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide-bovinethyroglobulin-conjugate

Step A

Bovine Thyroglobulin (BTG) Reaction with SATA:

To 3.0 mL of a solution of bovine thyroglobulin (BTG, 20.0 mg, 0.03μmoles) in 100 mM phosphate buffer pH 7.5 was added 276.0 μL of a DMFsolution of N-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 6.9 mg,30.0 μmoles). The resulting solution was incubated at 20° C. for 1 houron a roller mixer. The reaction was purified on a Sephadex G-25 columnusing 100 mM phosphate buffer, 5 mM EDTA, at pH 6.0. To 6.0 mL ofBTG-SATA (18.0 mg, 0.027 μmoles) was added 600 μL of 2.5 Mhydroxylamine, 50 mM EDTA, pH 7.0. The resulting solution was incubatedat 20° C. for 1 hour on a roller mixer.

Step B

To an aliquot of BTG-SH solution, prepared as described in the previousstep, 6.6 mL, 0.027 μmoles) was added an aliquot of the solutionprepared in Example 2 (898.9 μL, 19.0 μmoles). The resulting cloudymixture was incubated for 3 hours at 20° C. on a roller mixer. Thereaction was filtered through a 0.45 μm syringe filter, then purified ona Sephadex G-25 column using 100 mM phosphate buffer, 0.14M sodiumchloride, at pH 7.4.

Example 62-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((11-(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)dibenzo[b,f][1,4]thiazepin-3-yl)methyl)acetamide-bovinethyroglobulin-conjugate

To an aliquot of BTG-SH solution, prepared as described in Example 5Step A, (3.4 mL, 0.014 μmoles) was added 641.4 μL of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((11-(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)dibenzo[b,f][1,4]thiazepin-3-yl)methyl)acetamide,prepared as described in Example 4, (13.6 μmoles). The resulting cloudymixture was incubated for 3 hours at 20° C. on a roller mixer. Thereaction was purified on a Sephadex G-25 column using 100 mM phosphatebuffer, 0.14M sodium chloride, at pH 7.4.

Example 7N-((4-(dibenzo[b,f][1,4]thiazepin-11-yl)-1-(2-(2-hydroxyethoxy)ethyl)piperazin-2-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide-keyholelimpet hemocyanin-conjugate

Step A

Keyhole Limpet Hemocyanin (KLH) Reaction with SATA

To a 3.18 mL solution of keyhole limpet hemocyanin (KLH, 15.6 mg, 0.156μmoles) in 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4 wasadded 72.1 μL of a DMF solution of N-succinimidyl-S-acetylthioacetate(SATA, 25 mg/mL, 1.8 mg, 7.80 μmoles). The resulting solution wasincubated at 20° C. for 1 hour on a roller mixer. The reaction waspurified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46 Msodium chloride, 5 mM EDTA, at pH 6.0. To 6.27 mL of the resultingKLH-SATA solution (13.3 mg, 0.133 μmoles) was added 627 μL of 2.5Mhydroxylamine, 50 mM EDTA, at pH 7.0. The resulting solution wasincubated at 20° C. for 1 hour on a roller mixer. The reaction was usedas such in conjugation reaction with maleimide-activated hapten.

Step B

To an aliquot of KLH-SH solution, prepared as described in the previousstep, (6.9 mL, 0.133 μmoles) was added an aliquot of the solutionprepared in Example 2, (624.3 μL, 13.3 μmoles). The resulting cloudymixture was incubated for 3 hours at 20° C. on a roller mixer. Thereaction was filtered through a 0.45 μm syringe filter then purified ona Sephadex G-25 column using 100 mM phosphate buffer, 0.46M sodiumchloride, at pH 7.4.

Example 82-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((11-(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)dibenzo[b,f][1,4]thiazepin-3-yl)methyl)acetamide-keyholelimpet hemocyanin-conjugate

To an aliquot of the KLH-SH solution, prepared as described in Example 7Step A (3.2 mL, 0.061 μmoles) was added an aliquot of the solutionprepared in Example 4 (283.0 μL, 6.10 μmoles). The resulting cloudymixture was incubated for 3 hours at 20° C. on a roller mixer. Thereaction was purified on a Sephadex G-25 column using 100 mM phosphatebuffer, 0.46M sodium chloride, at pH 7.4.

Example 9

Competitive Immunoassays for Quetiapine and Multiplex CompetitiveImmunoassay for Aripiprazole, Olanzapine, Quetiapine, andRisperidone/Paliperidone

Following a series of immunizations with quetiapine immunogens, mousetail bleeds were tested for reactivity using an ELISA. Hybridomasupernatants were also tested, and the ELISA data shown in Tables 8 and9 below shows reactivity of several hybridomas (fusion partner was NSOcells).

TABLE 8 9 10 11 12 Dilution  400 79 89 90 95 Cmpd # 9  400 1200 12003600 3600 10800  10800  BI Sub 1.5858 1.3168 1.4302 0.0533 Cmpd # 91.5111 1.0627 1.2186 0.0427 0.5578 0.4213 0.598 0.0219 0.554 0.44470.5353 0.0233 0.1932 0.1582 0.1868 0.0154 0.171 0.2111 0.1838 0.01320.0736 0.0722 0.0733 0.0107 0.0884 0.0774 0.086 0.0107

TABLE 9 dilution 4C12 1A4 4G12 1F6  400 0.5467 0.2002 0.0144 0.1308 12000.1793 0.0619 0.01035 0.03905 3600 0.06655 0.026 0.00825 0.0192 10800 0.02755 0.0132 0.00765 0.01035  400 3.7296 0.24275 0.22585 0.00615 12002.4516 0.08695 0.0763 0.00685 3600 1.1575 0.0282 0.02875 0.00615 10800 0.4622 0.0147 0.0145 0.00645 dilution 5E9 2F2 3E2

Supernatant was then tested by competition ELISA to determine if thesignals were specific to quetiapine. FIGS. 1 and 2 show the results fromrepresentative hybridomas. Data shows specific reactivity to quetiapine.

FIG. 3 shows the competitive immunoassay format used on a lateral flowassay device in which the capture antibody, a quetiapine clone, wasdeposited on a chip along with a detection conjugate consisting ofquetiapine conjugated to a fluorophore. In this competitive format asshow in FIG. 3, a low level of analyte (quetiapine) results in highsignal, whereas a high level of analyte (quetiapine) results in lowsignal. The amount of quetiapine in the sample can be calculated fromthe loss of fluorescence compared to a control sample with no drugpresent. A typical dose response curve generated with quetiapinesub-clones 89-3, 89-13, and 89-5 is shown in FIG. 4.

FIG. 5 shows the chip design of a lateral flow assay device according toone embodiment of the subject invention. The device includes a zone orarea for receiving the sample, a conjugate zone (which contains desiredlabeled competitive binding partner(s)), and a reaction zone (eightareas within the reaction zone are indicated; each area can contain aseparate desired antibody). Sample flows from the sample zone throughthe conjugate zone and to the reaction zone.

FIGS. 6-9 show typical dose response curves for an aripiprazole positivecontrol (sample containing aripiprazole) generated with antibody 5C7deposited in reaction zone 2 and a labeled aripiprazole competitivebinding partner in the conjugate zone (FIG. 6), an olanzapine positivecontrol (sample containing olanzapine) generated with antibody 4G9-1deposited in reaction zone 4 and a labeled olanzapine competitivebinding partner in the conjugate zone (FIG. 7), a quetiapine positivecontrol (sample containing quetiapine) generated with antibody 11deposited in reaction zone 6 and a labeled quetiapine competitivebinding partner in the conjugate zone (FIG. 8), and a risperidonepositive control (sample containing risperidone) generated with antibody5-9 deposited in reaction zone 8 and a labeled risperidone competitivebinding partner in the conjugate zone (FIG. 9). The labeled competitivebinding partners in the conjugate zone compete with the drugs present inthe samples for binding to the antibodies. The amount of label isdetected and is an indication of the amount of drug present in thesample (the amount of signal being inversely proportional to the amountof drug in the sample—see FIG. 3).

In order to confirm that conjugates of labeled competitive bindingpartners do not bind to antibodies deposited in the reaction zones,negative controls were conducted by using samples containing no drugs.Referring to Table 10, a sample containing no aripiprazole is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled olanzapine, labeled quetiapine, andlabeled risperidone, but no labeled aripiprazole) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2. Table 10 below shows the results, confirming that thereis no dose response and the olanzapine, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the aripiprazole antibody.

TABLE 10 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) Re- PeakPeak Mean action Read Mean Mean Back- Assay-MM Conj Zone Position AreaHeight ground ARIP-MM1 OLAN, QUET, ARIP 2 0.77 1.56 3.99 RISP ARIP-MM1OLAN, QUET, 4 −0.02 0.06 4.14 RISP ARIP-MM1 OLAN, QUET, 6 0.09 0.10 4.29RISP ARIP-MM1 OLAN, QUET, 8 0.13 0.12 4.61 RISP Other Conjugates do notbind to Aripiprazole

Referring to Table 11, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled quetiapine, andlabeled risperidone, but no labeled olanzapine) and to the reactionzone. The reaction zone again contains olanzapine antibody (4G9-1) inreaction zone 4. Table 11 below shows the results, confirming that thereis no dose response and the aripiprazole, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the olanzapine antibody.

TABLE 11 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Re- Read PeakPeak Mean action Po- Mean Mean Back- Assay-MM Conj Zone sition AreaHeight ground OLAN-MM1 ARIP, QUET, 2 −0.03 0.05 4.38 RISP OLAN-MM1 ARIP,QUET, OLAN 4 0.74 1.10 4.56 RISP OLAN-MM1 ARIP, QUET, 6 0.06 0.09 4.79RISP OLAN-MM1 ARIP, QUET, 8 0.11 0.13 5.17 RISP Other Conjugates do notbind to Olanzapine

Referring to Table 12, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled olanzapine, andlabeled risperidone, but no labeled quetiapine) and to the reactionzone. The reaction zone again contains quetiapine antibody (11) inreaction zone 6. Table 12 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the quetiapine antibody.

TABLE 12 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Re- Read PeakPeak Mean action Po- Mean Mean Back- Assay-MM Conj Zone sition AreaHeight ground QUET-MM1 ARIP, OLAN, 2 −0.01 0.07 3.85 RISP QUET-MM1 ARIP,OLAN, 4 0.01 0.12 4.01 RISP QUET-MM1 ARIP, OLAN, QUET 6 0.03 0.08 4.24RISP QUET-MM1 ARIP, OLAN, 8 0.04 0.07 4.56 RISP Other Conjugates do notbind to Quetiapine

Referring to Table 13, a sample containing no risperidone is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled aripiprazole, labeled olanzapine, andlabeled quetiapine, but no labeled risperidone) and to the reactionzone. The reaction zone again contains risperidone antibody (5-9) inreaction zone 8. Table 13 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and quetiapineconjugates that move by capillary action through the reaction zone donot bind to the risperidone antibody.

TABLE 13 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) Re- PeakPeak Mean action Read Mean Mean Back- Assay-MM Conj Zone Position AreaHeight ground RISP-MM1 ARIP, OLAN, 2 0.02 0.11 7.43 QUET RISP-MM1 ARIP,OLAN, 4 0.05 0.14 7.73 QUET RISP-MM1 ARIP, OLAN, 6 0.20 0.19 8.11 QUETRISP-MM1 ARIP, OLAN, RISP 8 1.97 3.23 8.85 QUET Other Conjugates do notbind to Risperidone

In order to confirm that conjugates of labeled competitive bindingpartners bind only to their respective antibodies deposited in thereaction zones, additional negative controls were conducted by againusing samples containing no drugs. Referring to Table 14, a samplecontaining no aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (this time containinglabeled aripiprazole) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2, as well asolanzapine antibody (4G9-1) in reaction zone 4, quetiapine antibody (11)in reaction zone 6, and risperidone antibody (5-9) in reaction zone 8.Table 14 below shows the results, confirming that there is no doseresponse except to the aripiprazole antibody 5C7 (in reaction zone 2).

TABLE 14 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) Re- PeakPeak Mean action Read Mean Mean Back- Assay-MM Conj Zone Position AreaHeight ground ARIP-MM1 ARIP, OLAN, ARIP 2 60.34 97.53 5.44 QUET, RISPARIP-MM1 ARIP, OLAN, 4 2.86 3.91 11.66 QUET, RISP ARIP-MM1 ARIP, OLAN, 61.12 1.23 11.03 QUET, RISP ARIP-MM1 ARIP, OLAN, 8 3.14 4.19 12.94 QUET,RISP Only the Aripiprazole Reaction Zone is binding

Referring to Table 15, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled olanzapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 15 below shows the results, confirmingthat there is no dose response except to the olanzapine antibody 4G9-1(in reaction zone 4).

TABLE 15 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Re- Read PeakPeak Mean action Po- Mean Mean Back- Assay-MM Conj Zone sition AreaHeight ground OLAN-MM1 ARIP, OLAN, 2 0.02 0.08 4.86 QUET, RISP OLAN-MM1ARIP, OLAN, OLAN 4 34.23 51.80 5.39 QUET, RISP OLAN-MM1 ARIP, OLAN, 60.22 0.32 5.39 QUET, RISP OLAN-MM1 ARIP, OLAN, 8 0.15 0.17 5.59 QUET,RISP Only the Olanzapine Reaction Zone is binding

Referring to Table 16, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled quetiapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 16 below shows the results, confirmingthat there is no dose response except to the quetiapine antibody 11 (inreaction zone 6).

TABLE 16 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Re- Read PeakPeak Mean action Po- Mean Mean Back- Assay-MM Conj Zone sition AreaHeight ground QUET-MM1 ARIP, OLAN, 2 0.13 0.41 10.02 QUET, RISP QUET-MM1ARIP, OLAN, 4 0.08 0.23 10.47 QUET, RISP QUET-MM1 ARIP, OLAN, QUET 6140.35 181.33 7.91 QUET, RISP QUET-MM1 ARIP, OLAN, 8 1.58 2.61 11.53QUET, RISP Only the Quetiapine Reaction Zone is binding

Referring to Table 17, a sample containing no risperidone is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled risperidone) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2, as well as olanzapine antibody (4G9-1) in reaction zone4, quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 17 below shows the results, confirmingthat there is no dose response except to the risperidone antibody 5-9(in reaction zone 8).

TABLE 17 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) Re- PeakPeak Mean action Read Mean Mean Back- Assay-MM Conj Zone Position AreaHeight ground RISP-MM1 ARIP, OLAN, 2 1.03 1.51 9.07 QUET, RISP RISP-MM1ARIP, OLAN, 4 0.65 0.91 9.60 QUET, RISP RISP-MM1 ARIP, OLAN, 6 2.61 6.3910.48 QUET, RISP RISP-MM1 ARIP, OLAN, RISP 8 55.98 100.91 11.58 QUET,RISP Only the Risperidone Reaction Zone is binding

The results shown above confirm that conjugates of labeled competitivebinding partners bind only to their respective antibodies in thereaction zone.

FIGS. 10-13 show typical dose response curves in specific antibodyreaction zones, and proof of dose response low/high concentration foreach specific assay in the presence of other conjugates. In FIG. 10, asample containing aripiprazole is deposited in the sample zone and movesby capillary action through the conjugate zone (this time containinglabeled aripiprazole, labeled olanzapine, labeled quetiapine, andlabeled risperidone) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2. A typical doseresponse curve was generated as is shown in FIG. 10 only foraripiprazole, and not for olanzapine, quetiapine, or risperidone.

In FIG. 11, a sample containing olanzapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains olanzapine antibody (4G9-1) in reaction zone 4. A typicaldose response curve was generated as is shown in FIG. 11 only forolanzapine, and not for aripiprazole, quetiapine, or risperidone.

In FIG. 12, a sample containing quetiapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains quetiapine antibody (11) in reaction zone 6. A typicaldose response curve was generated as is shown in FIG. 12 only forquetiapine, and not for aripiprazole, olanzapine, or risperidone.

In FIG. 13, a sample containing risperidone is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains risperidone antibody (5-9) in reaction zone 8. A typicaldose response curve was generated as is shown in FIG. 13 only forrisperidone, and not for aripiprazole, olanzapine, or quetiapine.

FIGS. 14-17 show typical dose response curves for each assay in thepresence of other conjugates and antibodies. In FIG. 14, a samplecontaining aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (again containing labeledaripiprazole, labeled olanzapine, labeled quetiapine, and labeledrisperidone) and to the reaction zone. The reaction zone again containsaripiprazole antibody (5C7) in reaction zone 2, as well as olanzapineantibody (4G9-1) in reaction zone 4, quetiapine antibody (11) inreaction zone 6, and risperidone antibody (5-9) in reaction zone 8. Atypical dose response curve was generated for aripiprazole, as is shownin FIG. 14. When a sample containing olanzapine was deposited in thesample zone of this chip, a typical dose response curve was generatedfor olanzapine as shown in FIG. 15. When a sample containing quetiapinewas deposited in the sample zone of this chip, a typical dose responsecurve for quetiapine was generated as shown in FIG. 16. When a samplecontaining risperidone was deposited in the sample zone of this chip, atypical dose response curve for risperidone was generated as shown inFIG. 17.

FIGS. 18-21 show comparisons of dose response curves generated aspositive controls (FIGS. 6-9) to dose response curves generated in themultiplex format (FIGS. 14-17). The comparison for aripiprazole is shownin FIG. 18; for olanzapine in FIG. 19; for quetiapine in FIG. 20; andfor risperidone in FIG. 21. These figures show that the positive controlcurves are similar to the multiplex curves.

These data show that a lateral flow assay device of the subjectinvention can be used to detect multiple anti-psychotic drugs using asingle sample from a patient on one portable, point-of-care device.

What is claimed is:
 1. A method of producing an antibody or a bindingfragment thereof which specifically binds to quetiapine, the methodcomprising: (i) selecting a host for antibody production; and (ii)inoculating the host with a conjugate of a compound of Formula I and animmunogenic carrier; and (iii) isolating an antibody or binding fragmentthereof which specifically binds to quetiapine produced by the host,Formula I:

wherein: R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R³ is H; provided that either R¹ orR² must be H, and further provided that both R¹ and R² may not be Hsimultaneously; m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or 5, andwherein the immunogenic carrier is conjugated to the compound of FormulaI at R¹ when R¹ is not H, or at R² when R² is not H.
 2. A method ofproducing a hybridoma cell line capable of producing a monoclonalantibody which binds to quetiapine, the method comprising: (i) selectinga host for antibody production; (ii) inoculating the host with aconjugate of a compound of Formula I and an immunogenic carrier; (iii)fusing a cell line capable of producing an antibody that binds toquetiapine from said inoculated host with an immortalized cell to createa fused cell capable of producing a monoclonal antibody which binds toquetiapine; and (iv) cloning the fused cell so as to obtain a hybridomacell line, Formula I:

wherein:

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R³ is H; provided that either R¹ orR² must be H, and further provided that both R¹ and R² may not be Hsimultaneously; m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or 5, andwherein the immunogenic carrier is conjugated to the compound of FormulaI at R¹ when R¹ is not H, or at R² when R² is not H.
 3. The method ofclaim 1, wherein: R¹ is H,

or CH₂NH₂; R² is H,

or CH₂NH₂, R³ is H; provided that either R¹ or R² must be H, and furtherprovided that both R¹ and R² may not be H simultaneously; m is 1, 2, 3,4, or 5; and n is 1, 2, 3, 4, or
 5. 4. The method of claim 1, wherein R¹is H, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H, or CH₂NHC(O)(CH₂)_(m)CO₂H; R³is H; provided that either R¹ or R² must be H, and further provided thatboth R¹ and R² may not be H simultaneously; m is 1, 2, 3, 4, or 5; and nis 1, 2, 3, 4, or
 5. 5. The method of claim 1, wherein: R¹ is H,

or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

or CH₂NHC(O)(CH₂)_(m)CO₂H; R³ is H; provided that either R¹ or R² mustbe H, and further provided that R¹ and R² may not be H simultaneously; mis 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 6. The method of claim1, wherein the compound of Formula I is selected from the groupconsisting of:

wherein: m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 7. The methodof claim 2, wherein R¹ is H,

or CH₂NH₂; R² is H,

or CH₂NH₂, R³ is H; provided that either R¹ or R² must be H, and furtherprovided that both R¹ and R² may not be H simultaneously; m is 1, 2, 3,4, or 5; and n is 1, 2, 3, 4, or
 5. 8. The method of claim 2, wherein R¹is H, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H, or CH₂NHC(O)(CH₂)_(m)CO₂H; R³is H; provided that either R¹ or R² must be H, and further provided thatboth R¹ and R² may not be H simultaneously; m is 1, 2, 3, 4, or 5; and nis 1, 2, 3, 4, or
 5. 9. The method of claim 2, wherein: R¹ is H,

or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

or CH₂NHC(O)(CH₂)_(m)CO₂H; R³ is H; provided that either R¹ or R² mustbe H, and further provided that R¹ and R² may not be H simultaneously; mis 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 10. The method of claim2, wherein the compound of Formula I is selected from the groupconsisting of:

wherein: m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 11. Themethod of claim 1, wherein the immunogenic carrier is a protein.
 12. Themethod of claim 11, wherein the protein is selected from keyhole limpethemocyanin, bovine thyroglobulin, and ovalbumin.
 13. The method of claim2, wherein the immunogenic carrier is a protein.
 14. The method of claim13, wherein the protein is selected from keyhole limpet hemocyanin,bovine thyroglobulin, and ovalbumin.